Bottom Line:
A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring approximately 10 min after burning.This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids.Mass flow resumed 30-40 min after burning, as demonstrated by carboxyfluorescein movement and aphid activities.

ABSTRACTThe relationships between damage-induced electropotential waves (EPWs), sieve tube occlusion, and stop of mass flow were investigated in intact Cucurbita maxima plants. After burning leaf tips, EPWs propagating along the phloem of the main vein were recorded by extra- and intracellular microelectrodes. The respective EPW profiles (a steep hyperpolarization/depolarization peak followed by a prolonged hyperpolarization/depolarization) probably reflect merged action and variation potentials. A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring approximately 10 min after burning. Early stop of mass flow, well before completion of callose deposition, pointed to an occlusion mechanism preceding callose deposition. This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids. The early occlusion is probably due to proteins, as indicated by a dramatic drop in soluble sieve element proteins and a simultaneous coagulation of sieve element proteins shortly after the burning stimulus. Mass flow resumed 30-40 min after burning, as demonstrated by carboxyfluorescein movement and aphid activities. Stop of mass flow by Ca(2+)-dependent occlusion mechanisms is attributed to Ca(2+) influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca(2+) ions.

fig5: Distant protein plugging at the sieve plate after burning the leaf tip/major vein (at a distance of 9 cm from the observation window) as recorded using CLSM in intact Cucurbita maxima plants. The direction of mass flow is from the top (leaf tip) to the bottom (leaf base) of the picture. The bright spots are autofluorescent chloroplasts. Phloem tissue was pre-stained with 20 μM sulphorhodamine 101 for 20 min. (A) Control image at t=0 s showing the staining of plasma membranes. (B and C) Emergence of a fluorescent cloud (within the white circles) in the vicinity of the SP after 5 min and 30 min, respectively. SE, sieve element; CC, companion cell; SP, sieve plate.

Mentions:
The previous results (Fig. 3, 4) suggest ready occlusion of SPs at the time that callose deposition has hardly started. In analogy to the events in V. faba (Furch et al., 2007, 2009), proteins may be engaged in sieve tube occlusion. Therefore, the response of SE proteins to burning the leaf tip/major vein at a distance of 9 cm was observed using sulphorhodamine 101 (Fig. 5) and separation by 1-D SDS-PAGE (Fig. 6). Twenty minutes prior to burning, 10 μM sulphorhodamine 101 (cf. Peters et al., 2010), which preferentially associates with membranes, was applied onto the observation window. From 5 min after burning onwards, a cloud of fluorescence was observed at the SPs (Fig. 5B, C) indicative of protein clogging. It was technically impossible to further shorten the period between burning and observation.

fig5: Distant protein plugging at the sieve plate after burning the leaf tip/major vein (at a distance of 9 cm from the observation window) as recorded using CLSM in intact Cucurbita maxima plants. The direction of mass flow is from the top (leaf tip) to the bottom (leaf base) of the picture. The bright spots are autofluorescent chloroplasts. Phloem tissue was pre-stained with 20 μM sulphorhodamine 101 for 20 min. (A) Control image at t=0 s showing the staining of plasma membranes. (B and C) Emergence of a fluorescent cloud (within the white circles) in the vicinity of the SP after 5 min and 30 min, respectively. SE, sieve element; CC, companion cell; SP, sieve plate.

Mentions:
The previous results (Fig. 3, 4) suggest ready occlusion of SPs at the time that callose deposition has hardly started. In analogy to the events in V. faba (Furch et al., 2007, 2009), proteins may be engaged in sieve tube occlusion. Therefore, the response of SE proteins to burning the leaf tip/major vein at a distance of 9 cm was observed using sulphorhodamine 101 (Fig. 5) and separation by 1-D SDS-PAGE (Fig. 6). Twenty minutes prior to burning, 10 μM sulphorhodamine 101 (cf. Peters et al., 2010), which preferentially associates with membranes, was applied onto the observation window. From 5 min after burning onwards, a cloud of fluorescence was observed at the SPs (Fig. 5B, C) indicative of protein clogging. It was technically impossible to further shorten the period between burning and observation.

Bottom Line:
A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring approximately 10 min after burning.This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids.Mass flow resumed 30-40 min after burning, as demonstrated by carboxyfluorescein movement and aphid activities.

ABSTRACTThe relationships between damage-induced electropotential waves (EPWs), sieve tube occlusion, and stop of mass flow were investigated in intact Cucurbita maxima plants. After burning leaf tips, EPWs propagating along the phloem of the main vein were recorded by extra- and intracellular microelectrodes. The respective EPW profiles (a steep hyperpolarization/depolarization peak followed by a prolonged hyperpolarization/depolarization) probably reflect merged action and variation potentials. A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring approximately 10 min after burning. Early stop of mass flow, well before completion of callose deposition, pointed to an occlusion mechanism preceding callose deposition. This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids. The early occlusion is probably due to proteins, as indicated by a dramatic drop in soluble sieve element proteins and a simultaneous coagulation of sieve element proteins shortly after the burning stimulus. Mass flow resumed 30-40 min after burning, as demonstrated by carboxyfluorescein movement and aphid activities. Stop of mass flow by Ca(2+)-dependent occlusion mechanisms is attributed to Ca(2+) influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca(2+) ions.